Studies in animal models reveal that genetic differences and somatic mutations underlie longevity, but that non-genetic contributions also play a major role. Numerous observations, including our observations, suggest that epigenetic alterations occur as eukaryotes age. However, key questions remain, in particular, what are driving mechanisms and genomic changes that underlie the cellular phenotypes that characterize senescence and aging? Our hypothesis is that healthy aging involves homeostasis of the epigenomic landscape, which we refer to as chromostasis, and that chromostasis fails during aging, leading to tissue deterioration and to organismal death. Hence, genetic methods and pharmaco-therapeutics to enhance chromostasis are a prominent feature across all collaborative projects of this P01 application and in this Project 2. During the previous funding period we showed a key functional role of chromatin alterations in yeast replicative aging and massive chromatin alterations in mammalian senescence. In the next funding period we will explore and elucidate new chromatin regulatory pathways that alter genomic function in senescence and aging, leading to loss of chromostasis. In preliminary studies, we newly identified chromatin regulators whose reduction extends replicative lifespan, leading to new pathways that maintain epigenome and transcriptome fidelity during aging. We also discovered that nuclear disruption and shedding of LADs/chromatin into the cytoplasm during senescence and aging is perceived by a canonical cytoplasmic DNA sensing pathway, cGAS-STING, which in turn triggers cellular immunity pathways and the SASP (the senescence associated secretory phenotype) leading eventually to tissue damage during aging. To uncover the mechanisms and physiological importance of these new chromatin regulators and pathways in aging, we will carry out the following aims: 1. Investigate gene-internal cryptic transcriptional initiation during aging. We hypothesize that gene-internal transcriptional activation sites disrupt normal initiation at key longevity genes and lead to a global loss of transcriptional fidelity, contributing to reduction of chromostasis. (2) Investigate aging-associated upregulation of histone acetylation creating new enhancers. We hypothesize that dysregulated chromostasis licenses new enhancers during aging, leading to increased transcription of anti-longevity genes. (3) Investigate loss of chromatin integrity during aging triggering inflammation and autophagy of longevity chromatin regulators. Our preliminary findings show that LADs/chromatin in the cytoplasm triggers aging-promoting cellular immunity pathways via cGAS-STING. In the proposed studies, we will unravel the cGAS-STING pathway in promoting the SASP program in cellular senescence and the chronic inflammation associated with natural aging. This research will yield novel epigenetic mechanisms altering longevity, with potential for new therapeutic targets for intervention in age-related diseases and to extend healthy lifespan.